A variety of biosensors have been developed for the detection of biological material, such as pathogenic bacteria. Conventional methods for detecting bacteria usually involve a morphological evaluation of the organisms and rely on (or often require) growing the number of organisms needed for such an evaluation. Such methods are time consuming and are typically impractical under field conditions. The need for rapid detection as well as portability has led to the development of systems that couple pathogen recognition with signal transduction.
Requirements for an ideal detector include high specificity and high sensitivity using a protocol that can be completed in a relatively short time. Moreover, systems that can be miniaturized and automated offer a significant advantage over current technology, especially if detection is needed in the field.
The electrochemical methods use the principle of electrical circuit completion. To complete the electrical circuit, a counter electrode is used to provide a return path to the sample solution or reagent and a reference electrode is used as a reference point against which the potential of another electrode or electrodes are determined (typically that of the working electrode or measuring electrode). Since this contact must be provided by electrochemical means, i.e. a metal electrode immersed in a chemical solution or reagent, it is impossible to avoid generating an electrical potential in series with the potential developed by the electrode. The conventional theory in the electrochemical methods is that it is essential for the reference electrode potential to be very stable and not be affected by chemical changes in the solution. Thus, silver/silver chloride reference electrodes, which provide a very stable reference potential, are the most common type of electrode used for reference electrodes today.
Referring to FIG. 1, the typical silver/silver chloride reference electrode 10 contains a chloridised silver wire 1 (a layer of silver chloride coated on silver wire) immersed in a solution 5 of potassium chloride (3.5M KCl) saturated with silver chloride (AgCl). This internal filling solution 5 slowly seeps out of the electrode 10 through a porous ceramic junction 20 and acts as an electrical connection between the reference element 1 and the sample. Potassium chloride is used because it is inexpensive and does not normally interfere with the measurement. The solution 5 also includes silver chloride to prevent dissolution of the coating on the reference element 1. It is therefore necessary to maintain the level of solution 5 in the electrode 10 using a filling solution hole 40. This technique, however, is not robust and precise. In addition, it requires two-layered reference electrode (e.g., silver chloride coated on silver) having a known reference electrode potential described above.
Referring now to FIG. 2, the electrochemical methods also required a potentiostat 50, which is a control amplifier with the test cell placed in the feedback loop. The objective is to control the potential difference between a test electrode (working electrode) 60 and a reference electrode 70 by the application of a current via the third, auxiliary electrode (counter electrode) 80. In practice a fairly good potentiostat may be built using a minimum of components. In the circuit shown here, 90A and 90B are 1.22V bandgap reference diodes connected between the positive and negative power rails. Potentiometer 100 is then used to set the required cell polarization, applied to the non-inverting input of the main amplifier 110. The working electrode 60 connects to the ground of the circuit and the reference electrode 70 to the inverting input of the main amplifier 110. In order to boost the output capability somewhat (most operational amplifiers are limited to about 20 mA and do not tolerate capacitative loads well) a unity gain buffer amplifier 120 is used. Preferably, gain buffer amplifier 120 has a much higher bandwidth than amplifier 110, otherwise the circuit is likely to oscillate when driving a capacitative cell. The output of the buffer amplifier 120 connects to the auxiliary electrode 80 via a current measuring resistor 140. Differential amplifier 130 is then used to measure the voltage drop across this resistor 140 and to convert it to a ground referenced output voltage.
Microelectromechanical systems (MEMS) technology provides transducers to perform sensing and actuation in various engineering applications. The significance of MEMS technology is that it makes possible mechanical parts of micron size that can be integrated with electronics and batch fabricated in large quantities. MEMS devices are fabricated through the process of micromachining, a batch production process employing lithography. Micromachining relies heavily on the use of lithographic methods to create 3-dimensional structures using pre-designed resist patterns or masks. MEMS is one suitable technology for making microfabricated devices or aspects thereof. Microfabricated devices are generally defined as devices fabricated by using MEMS and/or integrated circuit (IC) technology. An IC is defined as a tiny chip of substrate material upon which is etched or imprinted a complex of electronic components and their interconnections. However, MEMS technology has not been successfully integrated with biosensing methods to detect various ionic molecules and macromolecules (DNA, RNA or protein), especially with electrochemocal methods, to provide biosensors with miniaturization and portability.
Existing techniques in biosensing of bacteria do not achieve high specificity and sensitivity with high dimensional precision. The detection is typically not applicable to a broad range of pathogenic bacteria. In addition, conventional biosensors are not miniaturized into a portable instrument.
Accordingly, there is a need to have a technique that can overcome the above disadvantages.